1. Field of the Invention
This invention generally relates to methods for monitoring the amount of antiplatelet compounds administered to a subject. More particularly, the invention provides methods and kits which allow for the rapid determination of the amount of an antiplatelet compound in a subject by the measurement of the activated clotting time of the subject's blood.
2. Description of the Prior Art
Platelets have a beneficial function in the cessation of blood flow (hemostasis) by providing the initial hemostatic plug at sites of vascular injury. Generally, the platelet first adheres to macromolecules in the subendothelial regions of an injured blood vessel and then aggregates to form the primary hemostatic plug. The aggregate of platelets near the injury then activates plasma coagulation factors which lead to the formation of a fibrin clot that supports and reinforces the aggregate.
Platelets also participate in harmful reactions such as those leading to atherosclerosis and pathological thrombosis. Thrombosis is a process in which a platelet aggregate and/or fibrin clot blocks a blood vessel. A thrombus blocking an artery may lead to the death of the tissue which is supplied blood by that artery. This blockage causes such conditions as stroke, unstable angina and myocardial infarction. Thrombosis can also cause complications after surgical procedures. For example, blood clots can form at sites which have been opened for implantation of prostheses, such as artificial heart valves, or for percutaneous transluminal angioplasty (PCTA). Antagonists of platelet function have therefore been studied and antiplatelet compounds developed to treat or prevent such complications arising from atherosclerosis and pathological thrombosis.
Many antiplatelet compounds having different functions are described in the art. Zablocki et al. describe fibrinogen receptor antagonists in development which function by disrupting the fibrinogen-platelet glycoprotein IIb/IIIa ("gpIIb/IIIa") interaction and are active inhibitors of all platelet activating agents. Zablocki, J. A. et al., Exp. Opin. Invest. Drugs, (1994) Vol. 3(5), pp. 437-448. Compounds such as dipyridamole, ticlopidine and aspirin are also known antiplatelet compounds. Majerus, P. W. et al. in Goodman and Gilman's The Pharmacological Basis of Therapeutics (Hardman, J. and Limbird, L. 9th Ed. 1996) pp. 1341-1359. Several patents covering antiplatelet compounds have issued and applications for patents covering additional compounds have been published. For example, U.S. Pat. No. 5,344,957 (Bovy et al.) discloses substituted .beta.-amino acid derivatives useful as platelet aggregation inhibitors and PCT Application Publication No. WO 94/22820 (Abood et al.) discloses 1-amidinophenyl-pyrrolidones, piperidinones and azetinones useful as platelet inhibitors. The disclosures of these references, patents and publications, including the references cited therein, are hereby incorporated into this specification to more fully define the state of the art.
Measurement of activated clotting times was developed by Hattersly as a sensitive test to monitor whole blood clotting. Hattersly, P. G., J.A.M.A., (1966) Vol. 196, pp. 150-154. Others have used the test as an assay to demonstrate drug activity. Moliterno et al. describe the increase of activated clotting times when the anti-gpIIb/IIIa antibody C7E3 is administered. Moliterno, D. et al. Am. J. Cardiol., (1995) Vol. 75, pp. 559-562.
As therapy using antiplatelet compounds increases, methods of monitoring the amount of the compound in the subject being treated becomes increasingly important. Monitoring concentrations, and adjusting doses in response thereto, will prevent overdosing and assist in tracking whether clearance of the compound may be impaired. Thus, a method which allows for a rapid and easy determination of the blood concentrations of these compounds is needed.
We have discovered that antibodies which immunoreact with an antiplatelet compound will affect the activated clotting time or platelet aggregation of blood containing that compound. The activated clotting time has been found to be significantly different in the presence of antibody such that an activated clotting time number, referenced herein as the "ACT number," can be calculated and correlated to a plotted curve or table giving specific concentrations of the compound in the blood, thus providing a rapid, non-labor intensive method of monitoring the concentration of these compounds.